The disclosed invention is in the general areas of bacteria with attenuated virulence and live bacterial vaccines.
The poxA gene (Van Dyk et al., J. Bacteriology169(10):4540-4546 (1987)), is a regulatory gene affecting expression of pyruvate oxidase (Chang and Cronan, J. Bacteriology 151(3): 1279-1289 (1982)). The poxA gene of E. coli is located at min 94. Enzymological and immunological data indicate that mutations in poxa have an 8 to 10-fold decrease in pyruvate oxidase levels (Chang and Cronan (1982); Chang and Cronan, J. Bacteriol. 154:756-762 (1983)). It has also been reported that poxA mutants grow more slowly than the isogenic wild-type in both minimal and rich media, while poxB mutants exhibit normal growth. Van Dyk and LaRossa, J. Bacteriol. 165(2):386-392 (1986), isolated 15 mutant Salmonella typhimurium strains sensitive to the herbicide sulfometuron methyl (SM) [N-[(4,6-dimethylpyrimidin-2-yl)aminocarbonyl]-2-methoxycarbonyl-benzenesulfonamide], following Tn10 mutagenesis. Among these SM-hypersensitive mutations, a poxA mutation was identified and mapped to the 94 min region of S. typhimurium genetic map (Van Dyk et al. (1987)), a location analogous to that of poxA in E. coli. The S. typhimurium poxA mutant, similarly to the E. coli poxA mutant, had reduced pyruvate oxidase activity and reduced growth rates (Van Dyk et al. (1987)). Furthermore, the E. coli and S. typhimurium poxA mutants shared several additional phenotypes including hypersensitivity to SM, to xcex1-ketobutyrate, and to a wide range of bacterial growth inhibitors, such as antibiotics, amino acid analogs and dyes (Van Dyk et al. (1987)).
The immune system of animals is especially suited to reacting to and eliminating microorganisms which infect the animal. The sustained presence of the full range of antigens expressed by infecting microorganisms provide a stimulating target for the immune system. It is likely that these characteristics lead to the superior efficacy, on average, of vaccines using live attenuated virus. For similar reasons, live bacterial vaccines have been developed that express a desired antigen and colonize the intestinal tract of animals (Curtiss et al., Curr. Topics Micro. Immun. 146:35-49 (1989); Curtiss, Attenuated Salmonella Strains as Live Vectors for the Expression of Foreign Antigens, in New Generation Vaccines (Woodrow and Levine, eds., Marcel Dekker, New York, 1990) pages 161-188; Schxc3x6del, Infection 20(1): 1-8 (1992); Cxc3xa1rdenas and Clements, Clinical Micro. Rev. 5(3):328-342 (1992)). Most work to date has used avirulent Salmonella typhimuriumstrains synthesizing various foreign antigens for immunization of mice, chickens and pigs. Several avirulent S. typhi vectors have been evaluated in human volunteers (Tacket et al., Infect. Immun.60:536-541 (1992)) and several phase I clinical trials with recombinant avirulent S. typhi strains are in progress in the U.S. and Europe. An important safety advantage of the live attenuated bacterial vaccine vectors as compared to the use of viral vector based vaccines is the ability to treat an immunized patient with oral ciprofloxacin or amoxicillin, should an adverse reaction occur.
It is understood that live bacteria administered to animals, and especially to humans, should not be pathogenic. Thus, live bacterial vaccines need to be avirulent. However, many bacteria that are most effective in stimulating an immune response, and would thus be most attractive as the subject of a live bacterial vaccine, are also the most virulent. Accordingly, there is a need for methods to reduce, or attenuate, the virulence of bacteria.
It is therefore an object of the present invention to provide a method of attenuating the virulence of a bacteria.
It is another object of the present invention to provide a method for inducing an immune response in an animal using bacterial cells with attenuated virulence.
It is another object of the present invention to provide compositions for inducing an immune response in an animal comprising bacterial cells with attenuated virulence.
It is another object of the present invention to provide an isolated nucleic acid molecule containing or encoding the poxR gene.
Disclosed are bacteria having virulence attenuated by a mutation to the regulatory gene poxR. Also disclosed is a method of producing bacteria having virulence attenuated by mutating the regulatory gene poxR. Such bacteria are useful for inducing an immune response in an animal or human against virulent forms of the bacteria with reduced risk of a virulent infection. Such bacteria are also useful to allow use of normally virulent bacteria as research tools with reduced risk of virulent infection. In a preferred embodiment, poxR attenuated bacteria can be used as a vaccine to induce immunoprotection in an animal or human against virulent forms of the bacteria. The disclosed bacteria can also be used as hosts for the expression of heterologous genes and proteins. Attenuated bacteria with such expression can be used, for example, to deliver and present heterologous antigens to the immune system of an animal. Such presentation on live bacteria can lead to improved stimulation of an immune response by the animal to the antigens.
The poxR gene, which is referred to in the literature as poxA (Van Dyk et al., J. Bacteriology 169(10):4540-4546 (1987)), is a regulatory gene affecting expression of pyruvate oxidase (Chang and Cronan, J. Bacteriology151(3):1279-1289 (1982)) and having other, pleiotropic effects. The pleiotropic phenotype effects include reduced pyruvate oxidase activity, reduced growth rate, hypersensitivity to the herbicide sulfometuron methyl, to xcex1-ketobutyrate and to amino acid analogs, and failure to grow in the presence of the host antimicrobial peptide, protamine. It has now been discovered that bacterial cell harboring a poxR mutation has significantly reduced virulence. Also disclosed is the nucleotide sequence of the poxR gene from Salmomella typhimurium, and the amino acid sequence of the encoded protein. The encoded protein has 325 amino acids and has significant sequence similarity to previously uncharacterized open reading frames in E. coli and Haemophilus influenzae.